Central to understanding how eukaryotic cells and the viruses that proliferate within them regulate the replication of their genomes is understanding the nature of DNA sites where replication begins and the proteins that interact with them. Previously, we and others have developed various strategies for mapping the locations of "origins of bidirectional replication" (OBRs) in the chromosomes of eukaryotic cells based on quantitative analyses of the amounts and distribution of nascent DNA labeled during its biosynthesis. These methods have been used by us as well as others to identify a number of replication origins in metazoan chromosomes to a resolution of #1 kilobase (kb), suggesting that metazoan chromosomes contain specific replication origins analogous to those found in the genomes of simpler organisms (e.g. yeast, protozoa). However, using 2D gel electrophoresis to detect structures of replication bubbles and forks, others have concluded that initiation events in metazoan cells occur randomly throughout "initiation zones" as large as 55 kb. During the past year, we have resolved much of this controversy by quantifying the relative number of early replication bubbles throughout the 55 kb initiation zone that lies between the hamster DHFR gene and the 2BE2121 gene. We were able to demonstrate the existence of two primary initiation sites (ori-b and b') that could account for the replication bubbles detected within the 12.2 kb region that gives the strongest initiation signals by 2D gel electrophoresis. These two OBRs, together with another one further downstream (ori-g) could account for most if not all of the initiation events within ~28 kb region in the center of this initiation zone. Therefore, initiation zones in mammalian cells, like those in fission yeast, consist of multiple primary initiation sites. We also have been able to show that some specific initiation sites, such as ori-b, depend upon the presence of specific DNA sequences that still retain their origin activity when translocated to ectopic chromosomal locations. These sequences do not contain an unusual "densely methylated island", as previously reported by another lab, although active ori-b loci are associated with a high density cluster of mCpG dinucleotides. Methylation is not required for initiation at ori-b, but it does appear to affect selection of ori-b over adjacent sequences. Initiation occurs at methylated ori-b sites in vivo and the resulting hemi-methylated daughter molecules are rapidly remethylated. However, while at least two origins are methylated (e.g. DHFR ori-b and rpS14), origins associated with promoters generally are not, even though they contain a high frequency of CpG dinucleotides (e.g. c-myc). Analysis of hypomethylated CHO cell lines and methylated plasmid DNAs suggests that while DNA methylation is not required for initiation of DNA replication, it strongly influences site specificity. In previous studies, we showed that site specific initiation can occur in a Xenopus egg extract when nuclei from G1-phase hamster cells are used as the substrate [Gilbert et al., Mol. Cell. Biol. 15: 2942-2954 (1995)]. However, the data indicated that these initiation events, while centered at ori-b/b', were also occuring less frequently at other sites in the genome. Changes in the protocol have now revealed that initiation events occur initially at ori-b and ori-b' (ori-g was not examined) and then at other sites later in the incubation, consistent with the existence of primary initiation sites within a genomic region that is generally accessible to initiation factors. Additional studies showed that initiation occurred at pre-replication complexes established in the hamster nucleus during late G1-phase of the cell division cycle. Therefore, changes must occur in chromatin structure or nuclear organization during G1-phase that repress assembly of pre-RCs at some sites while promoting their assembly at other sites. We have found that initiation sites for DNA replication in mammalian chromosomes are not defined by their association with nucleoskeleton, but so far all efforts to disassemble nuclear structure have resulted in loss of origin specificity. We have also found that neither DHFR nor 2BE2121 gene transcription correlates with assembly of specific initiation sites, and that potential RNA pol III promoters flanking ori-b are transcriptionally inactive, suggesting that origin specificity is not dependent on transcription in this region. Finally. we have cloned two of the mammalian homologues of yeast "origin recognition proteins" (ORC1, ORC2). ORC proteins are required to assemble pre-replication complexes in eukaryotes. These genes have been tagged and expressed in various systems. Antibodies are being made against these proteins and other proteins involved in assembly of pre-RCs such as Cdc6 and "mini-chromosome maintenance" (MCM) proteins in an effort to map the locations of these proteins within the DHFR gene initiation zone.